Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a food-grade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase.
Methods for the chemical synthesis of butanols are known. For example, 1-butanol may be produced using the Oxo process, the Reppe process, or the hydrogenation of crotonaldehyde (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719). 2-Butanol may be produced using n-butene hydration (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719). Additionally, isobutanol may be produced using Oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719) or Guerbet condensation of methanol with n-propanol (Carlini et al., J. Molec. Catal. A: Chem. 220:215-220 (2004)). These processes use starting materials derived from petrochemicals and are generally expensive and are not environmentally friendly.
Methods of producing butanol by fermentation are also known, where the most popular process produces a mixture of acetone, 1-butanol and ethanol and is referred to as the ABE processes (Blaschek et al., U.S. Pat. No. 6,358,717). Acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum is one of the oldest known industrial fermentations, and the pathways and genes responsible for the production of these solvents have been reported (Girbal et al., Trends in Biotechnology 16:11-16 (1998)). Additionally, recombinant microbial production hosts expressing a 1-butanol biosynthetic pathway (Donaldson et al., copending and commonly owned U.S. patent application Ser. No. 11/527,995), a 2-butanol biosynthetic pathway (Donaldson et al., copending and commonly owned U.S. Patent Application No. 60/796,816, and an isobutanol biosynthetic pathway (Maggio-Hall et al., copending and commonly owned U.S. patent application Ser. No. 11/586,315) have been described. However, biological production of butanols is believed to be limited by butanol toxicity to the host microorganism used in the fermentation.
Strains of Clostridium that are tolerant to 1-butanol have been isolated by chemical mutagenesis (Jain et al. U.S. Pat. No. 5,192,673; and Blaschek et al. U.S. Pat. No. 6,358,717), overexpression of certain classes of genes such as those that express stress response proteins (Papoutsakis et al. U.S. Pat. No. 6,960,465; and Tomas et al., Appl. Environ. Microbiol. 69(8):4951-4965 (2003)), and by serial enrichment (Quratulain et al., Folia Microbiologica (Prague) 40(5):467-471 (1995); and Soucaille et al., Current Microbiology 14(5):295-299 (1987)). Desmond et al. (Appl. Environ. Microbiol. 70(10):5929-5936 (2004)) report that overexpression of GroESL, a stress response protein, in Lactococcus lactis and Lactobacillus paracasei produced strains that were able to grow in the presence of 0.5% volume/volume (v/v) [0.4% weight/volume (w/v)] 1-butanol. Additionally, the isolation of 1-butanol tolerant strains from estuary sediment (Sardessai et al., Current Science 82(6):622-623 (2002)) and from activated sludge (Bieszkiewicz et al., Acta Microbiologica Polonica 36(3):259-265 (1987)) have been described. Additionally, some Lactobacillus species are known to be tolerant to ethanol (see for example, Couto et al., Biotechnol. Lett. 19:487-490 (1997) and Ingram et al. Adv. Microbial. Physiol. 25:253-300 (1984)). However, for most microorganisms described in the art, growth is totally inhibited at a concentration of less than 2.0% w/v 1-butanol when grown in a liquid medium at 37° C. Moreover, microbial strains that have a tolerance to 2-butanol and isobutanol are not known in the art. Therefore, microorganisms that have a high tolerance to 1-butanol, 2-butanol, and isobutanol would represent an advance in the art.
There is a need, therefore, for microbial host strains that are more tolerant to butanols and may be used for the bioproduction of butanols to high titer. The present invention addresses this need through the discovery of butanol tolerant microorganisms.
Applicants made the following biological deposits with an international depository (American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, USA) under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure:
InternationalDepositor IdentificationDepositoryReferenceDesignationDate of DepositPediococcus pentosaceusATCC: PTA-8068Dec. 7, 2006PN1011Pediococcus acidilactici PN1042ATCC: PTA-8069Dec. 7, 2006
The following sequences conform with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
TABLE 1Summary of Gene and Protein SEQ ID Numbersfor 1-Butanol Biosynthetic PathwaySEQ IDNO:SEQ IDNucleicNO:DescriptionacidPeptideAcetyl-CoA acetyltransferase thlA12from Clostridium acetobutylicumATCC 824Acetyl-CoA acetyltransferase thlB34from Clostridium acetobutylicumATCC 8243-Hydroxybutyryl-CoA56dehydrogenasefrom Clostridium acetobutylicumATCC 824Crotonase from Clostridium78acetobutylicum ATCC 824Putative trans-enoyl CoA910reductase from Clostridiumacetobutylicum ATCC 824Butyraldehyde dehydrogenase1112from Clostridium beijerinckiiNRRL B594Butanol dehydrogenase bdhB1314from Clostridium acetobutylicumATCC 824Butanol dehydrogenase1516bdhA from Clostridiumacetobutylicum ATCC 824
TABLE 2Summary of Gene and Protein SEQ ID Numbersfor 2-Butanol Biosynthetic PathwaySEQ IDNO:SEQ IDNucleicNO:DescriptionacidPeptidebudA, acetolactate decarboxylase1718from Klebsiella pneumoniaeATCC 25955budB, acetolactate synthase from1920Klebsiella pneumoniae ATCC25955budC, butanediol dehydrogenase2122from Klebsiella pneumoniaeIAM1063pddA, butanediol dehydratase2324alpha subunit from Klebsiellaoxytoca ATCC 8724pddB, butanediol dehydratase2526beta subunit from Klebsiellaoxytoca ATCC 8724pddC, butanediol dehydratase2728gamma subunit from Klebsiellaoxytoca ATCC 8724sadH, 2-butanol dehydrogenase2930from Rhodococcus ruber 219
TABLE 3Summary of Gene and Protein SEQ ID Numbersfor Isobutanol Biosynthetic PathwaySEQ IDNO:SEQ IDNucleicNO:DescriptionacidPeptideKlebsiella pneumoniae budB1920(acetolactate synthase)E. coli ilvC (acetohydroxy acid3132reductoisomerase)E. coli ilvD (acetohydroxy acid3334dehydratase)Lactococcus lactis kivD3536(branched-chain α-keto aciddecarboxylase), codon optimizedE. coli yqhD (branched-chain3738alcohol dehydrogenase)
SEQ ID NOs:39 and 40 are the nucleotide sequences of primers used to amplify the 16S rRNA genes of butanol tolerant strains, as described in Example 1.
SEQ ID NOs:41 and 42 are the nucleotide sequences of primers used to sequence the amplified 16S rRNA genes of butanol tolerant strains, as described in Example 1.
SEQ ID NOs:43 and 44 are the nucleotide sequences of the 16S rRNA genes of butanol tolerant Pediococcus strains, identified as described in Example 1.